CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation

The study addresses how tumor cells maintain proteostasis under stress and why resistance emerges to proteasome inhibitors (PIs) such as bortezomib. Proteostasis is crucial for cellular health and is often dysregulated in tumors due to extrinsic (hypoxia, nutrient deprivation) and intrinsic (oncogene activation, aneuploidy) stresses, creating “proteostasis addiction.” While PIs exploit this vulnerability and are effective in multiple myeloma and mantle cell lymphoma, resistance and limited efficacy in solid tumors remain challenges. CD317 (BST2/tetherin), a type II transmembrane glycoprotein implicated in viral restriction, inflammation, and tumor biology, is overexpressed in various cancers and activates signaling pathways that promote survival and drug resistance. Prior hints linked CD317 to ER stress pathways, suggesting a potential role in proteostasis regulation. The authors hypothesize that CD317 contributes to proteostasis maintenance in tumor cells, modulating ER calcium homeostasis and protein quality control, and thereby influences sensitivity to PIs.

Background literature establishes: (1) the centrality of ER proteostasis and the ubiquitin–proteasome system in cancer, with tumors often dependent on enhanced folding and degradation capacity; (2) clinical success of PIs in hematologic malignancies alongside issues of resistance; (3) CD317’s roles in cancer progression, signaling (EGFR, ERK, NF-κB), and resistance to therapies, and its involvement in ER stress responses in viral infection models; (4) calnexin (CNX) as an ER lectin chaperone that interacts with SERCA to restrain ER Ca2+ refilling and thereby modulate folding conditions; and (5) autophagy’s compensatory role when proteasomes are inhibited and the function of RACK1 as an autophagy regulator. This work builds on these findings to test whether CD317 regulates proteostasis via CNX and autophagy.

- Bioinformatics: Analyzed CD317 expression in hematologic malignancies using Oncomine datasets (Piccaluga lymphoma, Maia/Choi/Stegmaier leukemia, Zhan myeloma) and CCLE RNA-seq for cell lines. Survival correlations for CD317 and CNX in AML (LAML) assessed via OncoLnc (upper/lower percentiles 40%/60%). - Cell culture: HeLa, K562, Jurkat, NCI-H929 maintained in DMEM or RPMI-1640 with 10% FBS and L-glutamine; mycoplasma-free. - Gene perturbations: Transfected siRNAs targeting CD317, CNX, RACK1; siRNA-resistant CD317 constructs (full-length and domain mutants: delCT, delGPI). Overexpression plasmids for CNX (Myc) and RACK1 (Flag). Transfection via Lipofectamine 3000 or RFect. - Proteasome/autophagy modulators and reagents: Bortezomib (BTZ), MG-132, thapsigargin (TG), bafilomycin A1 (BAF), chloroquine (CQ), rapamycin, cycloheximide (CHX). Antibodies against CD317, CNX, CHOP, K48-linked ubiquitin, etc. - Calcium assays: Cytosolic Ca2+ measured using Fluo-4 AM by flow cytometry; ER Ca2+ stores inferred from TG-induced Ca2+ release dynamics; Fura-2 AM ratiometric imaging for complementary measurements. - Cell death: Annexin V/PI or PI staining by flow cytometry following BTZ treatment with/without gene knockdown or rescue. - qRT-PCR: Assessed mRNA levels of CD317, BiP, GRP94, HRD1. - Immunoblotting: Assessed K48-linked polyubiquitinated proteins (NP-40 soluble/insoluble fractions), CNX, CHOP, CD317, loading controls. - Co-immunoprecipitation (co-IP) and LC-MS/MS: Identified CD317 interactors; validated interactions between CD317–CNX and CD317–RACK1, and RACK1–CNX with/without CD317; domain mapping suggested requirement for CD317 GPI anchor. - Protein stability: Cycloheximide chase for CNX degradation kinetics in control vs CD317 knockdown. - Immunofluorescence/confocal microscopy: Co-localization of endogenous CD317 and CNX; CNX with LC3-labeled autophagosomes; analysis with line profiles and Pearson’s R. - Human specimens and IHC: Tissue microarrays (52 T-cell lymphoma, 8 normal lymphoid) stained for CD317 and CNX; semiquantitative scoring; correlation analysis. - RNA-seq and GSEA: Performed on K562 cells ± CD317 knockdown to assess pathway changes (KEGG; calcium signaling enrichment). - Statistics: Student’s t-test or Mann–Whitney U for two-group comparisons; Pearson correlation for IHC; log-rank test for survival; significance at P < 0.05. Experiments in triplicate; data as mean ± SEM.

- CD317 upregulation in hematologic malignancies: - Oncomine analyses show higher CD317 mRNA in BL, AML, MM compared to normals. - CCLE reveals elevated CD317 in multiple hematologic cancer cell lines. - IHC in T-cell lymphoma vs normal lymphoid tissue shows increased CD317 protein (P = 0.0219). - Higher CD317 expression correlates with worse overall survival in AML (OncoLnc; P = 0.0045). - Functional role in response to proteasome inhibition: - CD317 knockdown in K562, Jurkat, H929 does not affect basal proliferation/apoptosis but significantly increases BTZ-induced cell death; phenotype rescued by siRNA-resistant CD317. - Similar sensitization to BTZ observed in several solid tumor cell lines upon CD317 knockdown, reversed by CD317 rescue. - Proteostasis collapse upon CD317 loss: - Increased accumulation of K48-linked polyubiquitinated proteins (including NP-40–insoluble SDS-soluble fraction) after PIs with CD317 knockdown; rescued by CD317 re-expression. - Elevated ER stress markers: increased Bip, GRP94, HRD1 mRNAs and CHOP protein following PIs with CD317 knockdown. - Calcium homeostasis disruption: - GSEA indicates downregulation of calcium signaling upon CD317 knockdown (NES ≈ −1.203). - CD317 knockdown raises cytosolic Ca2+ and reduces ER Ca2+ stores across K562, Jurkat, H929; Fura-2 assays confirm; rescue by CD317 re-expression restores Ca2+ balance. - CD317–CNX interaction and CNX turnover: - LC-MS/MS identifies CNX as a CD317 interactor; co-IP validates endogenous and exogenous interactions; interaction enhanced by autophagy inhibition (BAF). - Confocal microscopy shows intracellular co-localization of CD317 and CNX. - CD317 knockdown increases CNX protein and slows CNX degradation (CHX chase). - In T-cell lymphoma tissues, CNX levels inversely correlate with CD317 (Pearson R = −0.3053, P = 0.0278). - RACK1-mediated autophagic degradation of CNX: - CD317 interacts with RACK1; interaction augmented by BAF; CD317’s GPI anchor required. - CD317 promotes RACK1–CNX interaction; loss of CD317 diminishes endogenous RACK1–CNX interaction. - Autophagy blockade (BAF) increases CNX and abrogates CNX differences between control and CD317 knockdown; rapamycin reduces CNX and nullifies CD317’s effect on CNX. - CD317 knockdown reduces autophagic flux; CD317-driven reduction of CNX requires RACK1 (RACK1 knockdown impairs CD317-mediated CNX degradation). - CNX is required for CD317’s effects on homeostasis and survival: - CNX knockdown in CD317-deficient cells restores ER and cytosolic Ca2+, reduces K48-polyubiquitin accumulation under BTZ, and rescues BTZ-induced cell death across K562, Jurkat, H929. - Mechanistic model: - CD317 scaffolds CNX to RACK1, promoting selective autophagic degradation of CNX; reduced CNX lifts inhibition on SERCA, enhancing ER Ca2+ refilling to support ER folding capacity and proteostasis; loss of CD317 elevates CNX, reduces ER Ca2+, triggers ER stress, and sensitizes cells to PIs.

The study demonstrates that CD317 is a critical regulator of tumor proteostasis by maintaining ER calcium homeostasis and supporting protein folding capacity. By engaging CNX and facilitating its RACK1-dependent autophagic degradation, CD317 fine-tunes SERCA activity and ER Ca2+ levels, which stabilizes the ER folding environment and mitigates proteotoxic stress. Consequently, CD317 loss leads to ER Ca2+ depletion, accumulation of misfolded proteins, heightened ER stress (including CHOP induction), and increased vulnerability to proteasome inhibition. These findings explain, mechanistically, how CD317 overexpression in hematologic malignancies can confer survival advantages under proteostasis stress and correlate with poor prognosis. The work also refines the understanding of autophagy in proteostasis, showing that beyond clearing misfolded proteins, autophagy actively optimizes the ER folding milieu via regulated turnover of CNX. Therapeutically, targeting CD317 could undermine this adaptive axis, potentially overcoming PI resistance or enhancing the efficacy of proteostasis-targeting regimens. Additionally, CNX levels may serve as a functional readout of CD317 activity and ER Ca2+ handling in tumors.

This work identifies CD317 as a previously unrecognized orchestrator of ER proteostasis in cancer cells. CD317 is upregulated in hematologic malignancies, correlates with poorer AML survival, and preserves cell viability during proteasome inhibition by promoting RACK1-mediated autophagic degradation of CNX, thereby sustaining ER Ca2+ homeostasis and protein folding. Loss of CD317 disrupts calcium balance, collapses proteostasis, and sensitizes tumor cells to PIs; reducing CNX reverses these effects. These findings propose CD317 as a therapeutic target to potentiate PI activity and address resistance. Future research should evaluate CD317-targeted strategies (e.g., antibodies, peptides, inhibitors) in vivo and clinically, define biomarkers (e.g., CNX, calcium signatures) predicting response, and explore combination therapies with PIs or autophagy modulators across malignancies, including solid tumors.